Peripheral arterial disease (PAD) is a major clinical problem afflicting 8-12 million Americans, and causes significant mortality and disability. Targeted medical or genetic therapies might provide clinical improvement of these patients through improved perfusion via vascular remodeling of existing collateral arteries (arteriogenesis) and/or by stimulating increased microvascular density (angiogenesis). There is an important interrelationship between these biological processes, however, it remains uncertain whether angiogenesis or arteriogenesis is the principal mediator of physiological improvement. We will characterize the interplay of these processes, in particular defining the role of avb3 integrins and nitric oxide (NO) in mediating hypoxia- induced angiogenesis and flow-induced arteriogenesis using well established murine models of hindlimb ischemia. The current proposal is a competitive renewal of a project previously directed at development of non-invasive radiotracer-based imaging strategies for identification of the hypoxic stimulus for angiogenesis, and direct evaluation of angiogenesis using radiotracers targeted at the avb3 integrin. A principal goal of the current project is to apply and refine these methodologies using non-invasive hybrid microSPECT/X-ray CT imaging strategies for the in vivo evaluation of the interdependent roles of angiogenesis and arteriogenesis in PAD, and to explore the role of NO in the regulation of integrins expressed on cells that mediate these processes. We hypothesize that ischemia may mediate the angiogenic process through regulation of integrins on several cell types, and that the resulting increases in tissue perfusion and decreases in vascular resistance result in increased flow-mediated arteriogenesis. The project will take advantage of established targeted radiotracers that selectively bind to the avb3 integrin, along with radiotracers that will track physiological changes in tissue perfusion and hypoxia associated with angiogenesis and arteriogenesis. The hybrid microSPECT/CT imaging system not only permits co-registration of biological images with developing vascular structures but facilitates quantification of tracer uptake. The current application builds directly on the novel imaging approaches previously developed, and will take advantage of established models of ischemia- and flow-induced angiogenesis to the study a critical mechanism of angiogenesis and arteriogenesis with potentially important clinical implications for evaluation and management of patients with PAD.